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Abstract:

The invention relates to systems and methods including a combination of
thermal generating device technologies to achieve more efficiency and
accuracy in PCR temperature cycling of nucleic samples undergoing
amplification.

Claims:

1. In a system comprising a nucleic acid sample, a heating device, and an
electromagnetic heating source, a method comprising: (a) controlling the
heating device to cause a temperature of the sample to be at or about a
first desired temperature for at least a first time period; (b) after
expiration of the first time period, increasing the output of the
electromagnetic heating source to cause the temperature of the sample to
be at or about a second desired temperature for at least a second time
period; (c) during said second time period, lowering the amount of heat
the heating device provides to the sample; and (d) immediately after
expiration of the second time period, lowering the output of the
electromagnetic heating source and controlling the heating device to
cause the temperature of the sample to be at or about a third desired
temperature for a third time period, wherein the first temperature is
less than the second temperature and the third temperature is less than
the first temperature.

2-27. (canceled)

Description:

[0001] This application claims the benefit of Provisional Patent
Application No. 60/806,440, filed on Jun. 30, 2006, which is incorporated
herein by this reference.

BACKGROUND

[0002] 1. Field of the Invention

[0003] The present invention relates to systems and methods for efficient
thermal cycling in DNA amplification using a combination of energy
sources, including electrical and/or magnetic (hereafter electromagnetic)
radiation as an energy source.

[0004] 2. Discussion of the Background

[0005] The detection of nucleic acids is central to medicine, forensic
science, industrial processing, crop and animal breeding, and many other
fields. The ability to detect disease conditions (e.g., cancer),
infectious organisms (e.g., HIV), genetic lineage, genetic markers, and
the like, is ubiquitous technology for disease diagnosis and prognosis,
marker assisted selection, identification of crime scene features, the
ability to propagate industrial organisms and many other techniques.
Determination of the integrity of a nucleic acid of interest can be
relevant to the pathology of an infection or cancer.

[0006] One of the most powerful and basic technologies to detect small
quantities of nucleic acids is to replicate some or all of a nucleic acid
sequence many times, and then analyze the amplification products.
Polymerase chain reaction (PCR) is a well-known technique for amplifying
DNA. With PCR, one can produce millions of copies of DNA starting from a
single template DNA molecule. PCR includes phases of "denaturation,"
"annealing," and "extension." These phases are part of a cycle which is
repeated a number of times so that at the end of the process there are
enough copies to be detected and analyzed. For general details concerning
PCR, see Sambrook and Russell, Molecular Cloning--A Laboratory Manual
(3rd Ed.), Vols. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor,
N.Y. (2000); Current Protocols in Molecular Biology, F. M. Ausubel et
al., eds., Current Protocols, a joint venture between Greene Publishing
Associates, Inc. and John Wiley & Sons, Inc., (supplemented through 2005)
and PCR Protocols A Guide to Methods and Applications, M. A. Innis et
al., eds., Academic Press Inc. San Diego, Calif. (1990).

[0007] The PCR process phases of denaturing, annealing, and extension
occur at different temperatures and cause target DNA molecule samples to
replicate themselves. Temperature cycling (thermocyling) requirements
vary with particular nucleic acid samples and assays. In the denaturing
phase, a double stranded DNA (dsDNA) is thermally separated into single
stranded DNA (ssDNA). During the annealing phase, primers are attached to
the single stand DNA molecules. Single strand DNA molecules grow to
double stranded DNA again in the extension phase through specific
bindings between nucleotides in the PCR solution and the single strand
DNA. Typical temperatures are 95° C. for denaturing, 55° C.
for annealing, and 72° C. for extension. The temperature is held
at each phase for a certain amount of time which may be a fraction of a
second up to a few tens of seconds. The DNA is doubled at each cycle; it
generally takes 20 to 40 cycles to produce enough DNA for the
applications. To have good yield of target product, one has to accurately
control the sample temperatures at the different phases to a specified
degree.

[0008] More recently, a number of high throughput approaches to performing
PCR and other amplification reactions have been developed, e.g.,
involving amplification reactions in microfluidic devices, as well as
methods for detecting and analyzing amplified nucleic acids in or on the
devices. Thermal cycling of the sample for amplification is usually
accomplished in one of two methods. In the first method, the sample
solution is loaded into the device and the temperature is cycled in time,
much like a conventional PCR instrument. In the second method, the sample
solution is pumped continuously through spatially varying temperature
zones. See, for example, Lagally et al. (Analytical Chemistry 73:565-570
(2001)), Kopp et al. (Science 280:1046-1048 (1998)), Park et al.
(Analytical Chemistry 75:6029-6033 (2003)), Hahn et al. (WO 2005/075683),
Enzelberger et al. (U.S. Pat. No. 6,960,437) and Knapp et al. (U.S.
Patent Application Publication No. 2005/0042639).

[0009] Many detection methods require a determined large number of copies
(millions, for example) of the original DNA molecule, in order for the
DNA to be characterized. Because the total number of cycles is fixed with
respect to the number of desired copies, the only way to reduce the
process time is to reduce the length of a cycle. Thus, the total process
time may be significantly reduced by rapidly heating and cooling samples
to process phase temperatures while accurately maintaining those
temperatures for the process phase duration.

[0010] Accordingly, what is desired is a system and method for rapidly and
accurately changing process temperatures in PCR processes.

[0012] In one aspect, the present invention provides a method for cycling
the temperature of a nucleic acid sample. In one embodiment, the method
includes: (a) controlling a heating device to cause a temperature of the
sample to be at or about a first desired temperature for at least a first
time period; (b) after expiration of the first time period, increasing
the output of an electromagnetic heating source to cause the temperature
of the sample to be at or about a second desired temperature for at least
a second time period; (c) during said second time period, lowering the
amount of heat the heating device provides to the sample; and (d)
immediately after expiration of the second time period, lowering the
output of the electromagnetic heating source and controlling the heating
device to cause the temperature of the sample to be at or about a third
desired temperature for a third time period, wherein the first
temperature is less than the second temperature and the third temperature
is less than the first temperature. In some embodiments, steps (a)
through (d) occur while the sample is flowing through a channel (e.g., a
microfluidic channel).

[0013] In another embodiment, the method includes: heating the sample to a
first temperature for a first time period using a thermoelectric device;
heating the sample to a second temperature for a second time period using
primarily an electromagnetic heat source; cooling the sample to a third
temperature; and maintaining the third temperature for a third time
period using the thermoelectric device, wherein the second temperature is
higher than the first and the first temperature is higher than the third.

[0014] In another embodiment, the method includes: (a) heating the nucleic
acid sample to about a first temperature; (b) after heating the sample to
about the first temperature, maintaining the temperature of the sample at
about the first temperature for a first period of time; (c) after
expiration of the first period of time, heating the sample to about a
second temperature; (d) after heating the sample to the second
temperature, maintaining the temperature of the sample at about the
second temperature for a second period of time; (e) after expiration of
the second period of time, cooling the sample to about a third
temperature; and (f) after cooling the sample to the third temperature,
maintaining the temperature of the sample at about the third temperature
for a third period of time, wherein the first temperature is less than
the second temperature and greater than the third temperature, and the
step of heating the sample to the second temperature consists primarily
of using one or more non-contact heating elements to heat the sample to
the second temperature.

[0015] In another aspect, the present invention provides a system for
cycling the temperature of a nucleic acid sample. In one embodiment, the
system includes: a nucleic acid sample container operable to receive a
nucleic acid sample; a first heating device; and a second heating device,
wherein the first heating device is configured to heat the nucleic acid
sample to at least about a first temperature, the second heating device
is configured to heat the nucleic acid sample to a second and third
temperature, the first temperature is associated with a denaturing phase
of a PCR process, the first heating device is a non-contact heating
device, and the second heating device is a contact heating device.

[0016] The above and other embodiments of the present invention are
described below with reference to the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The accompanying drawings, which are incorporated herein and form
part of the specification, illustrate various embodiments of the present
invention. In the drawings, like reference numbers indicate identical or
functionally similar elements. Additionally, the left-most digit(s) of a
reference number identifies the drawing in which the reference number
first appears.

[0018]FIG. 1 depicts an apparatus in accordance with an exemplary
embodiment of the invention.

[0020]FIG. 3 depicts a temperature characteristic of a heating device and
of an electromagnetic heating device.

[0021] FIGS. 4 and 5 depict a temperature cycle.

[0022] FIG. 6 depicts steps in an exemplary method in accordance with an
embodiment of the present invention.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

[0023] With reference to FIG. 1, an exemplary embodiment of an apparatus
100 relating to the present invention may include: a microfluidic device
101 or other device for containing a sample containing a nucleic acid and
PCR reagents (which PCR reagents may include PCR primers, dNTPs,
polymerase enzymes, salts, buffers, surface-passivating agents, and the
like), a heat spreader 102, a heating device 103, a heat sink 104, an
electromagnetic heat source 105, a contact temperature sensing device
107, a non-contact temperature sensing device 108, a controller 109 and a
fan 110.

[0024] In some embodiments, device 101 includes a microfluidic channel
configured to receive the sample. The sample may flow through the channel
as its temperature is cycled, as described herein. Moving the sample
through the microfluidic channel can be accomplished by a variety of
methods, for example, via conventional methods of pressure-driven flow
(e.g., using a pump to create a pressure differential) and the flow rates
can vary, for example between 10 nanoliters per minute to 1 ml per
minute.

[0025] In the embodiment illustrated, heating device 103 is thermally in
contact with the microfluidic device 101 through the metal heater
spreader 102. Device 103 may be implemented using a thermoelectric cooler
(TEC) (also referred to as a Peltier device), a resistive heater, a
chemical heater, or other heating device. A suitable TEC may be available
from Melcor Corporation of Trenton, N.J. (see part number HT6-6-21X43).
In some embodiments, heating device 103 may have a different temperature
resolution than heat source 105. More specifically, in some embodiments,
heating device 103 may have a finer temperature resolution than heat
source 105.

[0026] As mentioned above, device 103 may be implemented using a Peltier
device, a widely used component in laboratory instrumentation and
equipment, well known among those familiar with such equipment, and
readily available from commercial suppliers such as Melcor. A Peltier
device is a solid-state device that can function as a heat pump, such
that when an electric current flows through two dissimilar conductors,
the junction of the two conductors will either absorb or release heat
depending on the direction of current flow. A typical Peltier device
consists of two ceramic or metallic plates separated by a semiconductor
material, such as bismuth telluride. The direction of heat flow is a
function of the direction of the electric current and the nature of the
charge carrier in the semiconductor (i.e., n-type or p-type). Peltier
devices can be arranged and/or electrically connected in an apparatus of
the present invention to heat or to cool a PCR process taking place in
microfluidic device 101.

[0027] The size of heat spreader 102 is related to the sizes of the
heating device 103 and microfluidic device 101. In an exemplary
embodiment, a heat spreader of 20 mm×40 mm is used. Heat sink 104
removes waste heat from heating device 103. The assembly comprising the
heat sink 104, heating device 103, and heater spreader 102 may be screwed
together, bonded together, or clamped in place. Thermal coupling may be
enhanced by the use of thermally conductive adhesives, greases, pastes,
thermoconducting pads (e.g., a SIL-PAD product available from the
Berquist Company of Chanhassen, Minn.).

[0028] A switchable fan 110 may be used to increase airflow towards the
assembly and/or heat sink 104 in particular. The fan 110 and heat sink
104 can function together to quickly remove heat from the assembly. In
some embodiments, fan 110 may be left on continuously during for an
entire PCR process.

[0029] Electromagnetic heat source 105 may radiate energy 106 directed
toward the microfluidic device 101 surface. A suitable electromagnetic
heat source is any device which generates an electric and/or magnetic
field which may be used to heat microfluidic device 101. An exemplary
electromagnetic heat source 105 may be an infrared source including a
tungsten filament bulb, such as one from the GE XR series, or a laser.
Heat source 105 may be located 1/2 of an inch or less to twelve inches or
more from the surface of microfluidic device 101. Preferably, heat source
105 is located at an angle so as to facilitate real-time PCR monitoring
of the microfluidic zone and is located between about 2-6 inches from the
surface of device 101.

[0030] Contact temperature sensing device 107 may be located inside or on
the microfluidic device 101 surface. Suitable temperature sensors include
a thin film wire, embedded wire, thermocouple, RTD, resistor, or solid
state device. In some embodiments, a temperature measuring sensor
available from Analog Devices is used to implement device 107 (e.g., the
Analog Devices AD590 temperature transducer may be used). In some
embodiments, a non-contact temperature sensing device 108, such as a
pyrometer manufactured by Mikron, may be used in addition to or instead
of contact temperature sensing device 107.

[0031] Controller 109 may be used to energize and deenergize heating
device 103 and electromagnetic heat source 105 in a thermostatic fashion
such that the temperature sensed by sensor 107 and/or 108 (e.g., the
temperature of a region of microfluidic device 101) is at, or
approximately at, a desired temperature for a desired period of time.
Controller 109 include one or more computers or other programmable
devices which may be programmed to control heating device 103,
electromagnetic heat source 105, and/or fan 111 in response to the
expiration of a timer and temperature measurements from contact
temperature sensor 107 and/or non-contact temperature sensor 108.

[0032]FIG. 2 depicts an exemplary desired PCR cycle. In this exemplary
cycle, phase 211 is the extension phase, phase 212 is the denaturation
phase, and phase 213 is the annealing phase. Typically, the denaturation
phase 212 requires less precision of temperature and time control,
provided that a minimum temperature point is achieved homogeneously in
the microfluidic device 101.

[0033] In a preferred embodiment, controller 109 is programmed to use
primarily heating device 103 to heat and/or cool microfluidic device 101
during extension phase 211 and annealing phase 213. That is, in some
embodiments, source 105 may be turned "off" during the extension phase
and annealing phase or may output a lower level of radiation 106 during
these phases than it outputs during denaturation phase 212. Controller
109 is operable to energize the heating and/or cooling ability of heating
device 103 such that the desired temperatures are quickly reached and
maintained for the desired times. For example, an extension phase 211 may
have a duration of about 5 seconds and a desired temperature of about
72° C. An exemplary annealing phase 213 may have a desired
duration of 2 seconds and a desired temperature of about 55° C.

[0034] In the preferred embodiment, electromagnetic heat source 105
provides to microfluidic device 101 a greater amount of heat during the
denaturation phase 212 than during the other two phases of the PCR cycle.
Additionally, during denaturation phase 212, device 103 may be operated
to provide less heat to device 101 than device 103 is configured to
provide to device 101 during the other two phases of the PCR cycle.
Accordingly, in some embodiments, device 103 may actually draw heat from
device 101 during denaturation phase 212. An exemplary desired
denaturation phase 212 may last about 500 ms at a desired temperature of
about 95° C.

[0035]FIG. 3 depicts exemplary plots of a heating device 103 temperature
characteristic 301 and source 105 temperature characteristic 303.
Characteristic 301 illustrates that the heating device 103 is controlled
by the controller 109 such that the desired temperature control of the
sample for the extension phase 211 and annealing phase 213 is
substantially provided through the functioning of the heating device 103.
Characteristic 303 illustrates that the electromagnetic heat source 105
is controlled by the controller 109 such that the desired temperature
control of the sample for denaturation phase 212 is substantially
provided through the functioning of the electromagnetic heat source 105.

[0036] FIGS. 4 and 5 depict time and temperature graphs for a sample
contained in device 101 when heat sources 103 and 105 are operated
according to the diagram shown in FIG. 3. Shaded area 441 represents the
part of the cycle for which device 103 provides thermal input. Period A,
which occurs in area 440, represents the duration of an energy input from
source 105.

[0037] Accordingly, and with reference to FIG. 5, fast thermal ramp/rise
rates may be achieved as indicated by slope 530. Furthermore, high
cooling rates may be achieved as indicated by slope 531.

[0038] Referring now to FIG. 6, FIG. 6 is a flow chart illustrating a
process, according to some embodiments of the invention, for cycling the
temperature of a sample that is present in device 101.

[0039] The process may begin in step S601, where the controller controls
thermostatically heating device 103 such that the extension phase
temperature is reached and maintained for a desired duration of the
extension phase 211. While step S601 is being performed, source 105 may
be in an "off" state.

[0040] In step S603, which preferably does not occur until about
immediately after the expiration of the desired extension phase duration,
controller 109 increases the output of electromagnetic heat source 105.
Preferably, the output of source 105 is raised to a level that causes the
temperature of the sample to rapidly increase to the denaturation phase
temperature.

[0041] In step 605, controller 109 may control thermostatically one or
more of the heating and cooling devices of apparatus 100 so that the
sample is kept at the denaturation phase temperature for the desired
duration of the denaturation phase 212.

[0042] At or about the same time controller 109 causes heat source 105 to
heat the sample to the denature temperature, controller 109 may control
device 103 such that the heat provided by device 103 to device 101 is
less than the heat device 103 provided to device 101 during step S601
(i.e., during extension phase 211).

[0043] In step S607, which preferably does not occur until about
immediately after the expiration of the desired denature phase duration,
controller 109 lowers the energy output (e.g., turns off) electromagnetic
heat source 105 and controls heating device 103 such that the annealing
phase temperature is reached. In some embodiments, step S607 includes
using the cooling capability of the heating device to quickly drive the
temperature of the sample down from the denaturation phase temperature to
the annealing phase temperature as shown in ramp 531. In some
embodiments, step S607 also includes activating and directing fan 110 at
the microfluidic device 101 and/or heat sink 104 to drive the temperature
down rapidly as shown in ramp 631.

[0044] In step S609, controller 109 may control thermostatically one or
more of the heating and cooling devices of apparatus 100 so that the
sample is kept at the annealing phase temperature for the desired
duration of the annealing phase 213. For example, if a temperature sensor
(e.g., sensor 107 or 108) indicates that the temperature of the sample is
too low, then controller 109 may control a heat source (e.g., device 103
or source 105) to add more heat to the sample, and if a temperature
sensor 108 indicates that the temperature of the sample is too low, then
controller 109 may control device 103 so that it draws heat from the
sample.

[0045] Embodiments of the present invention have been fully described
above with reference to the drawing figures. Although the invention has
been described based upon these preferred embodiments, it would be
apparent to those of skill in the art that certain modifications,
variations, and alternative constructions could be made to the described
embodiments within the spirit and scope of the invention.

[0046] Furthermore, one of skill in the art will recognize that
temperatures enumerated in the following claims should be interpreted to
mean "at or about" the enumerated temperature.

[0047] For the claims below the words "a" and "an" should be construed as
"one or more."

Patent applications by Gregory A. Dale, Gaithersburg, MD US

Patent applications by Kenton C. Hasson, Germantown, MD US

Patent applications by Shulin Zeng, Gaithersburg, MD US

Patent applications by Canon U.S. Life Sciences, Inc.

Patent applications in class Including condition or time responsive control means

Patent applications in all subclasses Including condition or time responsive control means